The present invention relates to a process for treating hypertension through the administration of an effective amount of a an inhibitor to the renin-angiotensin system of a mammal in order to inhibit the formation of the heptapeptide Angiotensin-(1-7). Examples of such inhibitors include Z-Pro-prolinal, N-{N-[1-(S)-carboxy-3-phenylpropyl]-(S)-phenylalanyl}-(S)-isoserine (SCH 39,370), and carboxy-phenylpropylalanine- phenylalanine-para-aminobenzoate (cFP-A-A-F-pAB). In addition, the present invention further relates to a pharmaceutical composition for treating hypertension in mammals comprised of an Angiotensin-(1-7) inhibitor as the active ingredient. In this regard, inhibitors such as Z-Pro prolinal have recently been discovered by the present inventors inhibit the formation of the heptapeptide Angiotensin-(1-7) [Ang-(1-7)], an N-terminal fragment of Angiotensin II (Ang II) previously considered to be an inert product of the renin-angiotensin system (RAS). Through the administration of an inhibitor such as an effective amount of a Z-Pro-prolinal (ZPP) composition, Ang-(1-7) formation is inhibited and the blood pressure in mammals can be controlled without notable changes in heart rate and other circulatory functions.
The renin-angiotensin system (RAS) is an important regulatory system for controlling blood pressure in mammals. Although the voluminous literature on the biochemical pathways and physiological actions of the renin-angiotensin system (RAS) contains notable controversies, it has never been questioned that the octapeptide Angiotensin II (Ang II) is the biologically active principle of the RAS. In this regard, it has generally been assumed that the interaction of Ang II with specific receptors found on several organs produces the systematic effects related to the control of blood pressure. The most prominent of the effects produced by circulating Ang II is the direct vasoconstriction of the peripheral vasculature, which is normally accompanied by a variety of changes in kidney functions such as alterations in the glomerular filtration rate, tubular reabsorption, and renal arteriolar resistance.
Moreover, not only is Ang II's function in the peripheral tissue as a plasma hormone well known, its biosynthetic pathway for formation has also been well documented. The following steps are generally stated to be the pertinent steps in the formation of Ang II in the RAS: ##STR1##
The production of Ang II from the polypeptide precursor, Angiotensinogen, is regulated by two proteolytic enzymes, renin and angiotensin converting enzyme (ACE), which cleave successively at the Leu.sup.10 -Val.sup.11 and Phe.sup.8 -His.sup.9 bonds to produce Angiotensin I (Ang I) and Ang II, respectively. Renin occurs predominantly in the juxtaglomerular cells of the kidney but has also been detected at a number of extrarenal sites. ACE, a dipeptidyl-carboxypeptidase, is normally present in the serum; the endothelial cells of the pulmonary vascular bed; and also in many other tissues such as the kidney, gut, brain, and testis. The combined efforts of the kidney to produce renin when a decrease in blood pressure is detected, of the liver to produce the renin substrate, Angiotensinogen (Aogen), found in the circulating blood (which is cleaved by the renin to form Ang I) and of the lungs to produce the ACE (which converts the Ang I to Ang II) are all required for the generation of the biologically active peptide Ang II in the peripheral tissues.
The generated Ang II in turn increases blood pressure by constricting blood vessels and by activating aldosterone secretion which stimulates sodium retention (and potassium wasting) by the renal tubule. In addition, it is also thought that Ang II induces thirst and arginine vasopressin (AVP) release in the systemic system.
Furthermore, while renin and ACE play critical roles in the processing of Angiotensinogen (Aogen) to Ang II, other endo- and carboxypeptidases are thought to contribute as well to the formation of Ang II and/or Ang II analogs such as Ang-(2-8), Ang-(1-7), etc. Studies of the in vivo and in vitro catabolism of Angiotensin peptide precursors, and of Ang II in tissues, suggest that some of the C-terminal fragments formed through metabolism of Ang I and Ang II may be bioactive.
In addition, although other angiotensin fragments and/or analogs of Ang II derived from the aminoterminus (N-), such as Ang-(1-7), have been isolated and purified (i.e. enzymes which cleave peptides at the Proline-Phenylalanine bond exist in many tissues), it has generally been concluded from studies of the structure-activity relationship in Ang II analogs, that fragments of Ang II lacking an aminoacid residue in position 8 of the polypeptide are inert. Along the same lines, it is widely believed in the art that the phenyl group in position 8 contains the information necessary for regulating blood pressure in mammals.
In this regard, while Yang, et al. first showed the generation of an N-terminal fragment of Ang II [the heptapeptide Angiotensin-(1-7)] in swine and human urine (Yang, H. Y. T., Erdos, E. G., and Chang, T. S., New Enzymatic Route for the Inactivation of Angiotensin, Nature 218: 1224-1226, 1968), and similar findings were obtained by Regoli, et al. in vascular smooth muscle (Regoli, D., Park, W. K., Rioux, F., and Magnan, J., Metabolism of Angiotensin in Vascular Smooth Muscle: In Biologically Active Peptides. R. Waher & J Meienh, (eds.). Ann Arbor, Mich.; Ann Arbor Sci. Publishers, Inc., pp: 617-624, 1975), by Tonnaer, et al. in preparations of synaptic membranes from the rat brain (Tonnaer, J. A., Wiegant, V. M., DeJong, W., and DeWied, D., Central Effects of Angiotensins on Drinking and Blood Pressure: Structure--Activity Relationships, Brain Res. 236: 417-428, 1982; and, Tonnaer, J. A., Engles, G. M., Wiegant, V. M., Burbach, J. P., DeJong, W., and Diewied, D., Proteolytic Conversion of Angiotensins in Rat Brain Tissue, Eur. J. Biochem., 131: 415-421, 1983), and by Allard, et al. in cultured mouse spinal cord cells (Allard, M., Simonnet, G., Dupouy, B., and Vincent, J. D., Angiotensin II Inactivation Process in Cultured Mouse Spinal Cord Cells, J. Neurochem. 48: 1553-1559, 1987), pharmacological studies showed that the Ang-(1-7) did not elicit contractile responses in isolated vessels and/or demonstrate the pressor, dipsogenic, or aldosterone stimulating properties of Ang II. From these studies it was concluded that fragments of Ang II derived from the amino terminus (N-), such as Ang-(1-7), have no biological activity.
Moreover, although it has been generally assumed that the RAS maintains blood pressure through Ang II generated in the circulation, recent evidence clearly indicates the existence of a separate renin-angiotensin system (RAS) in the brain of mammals (i.e. brain RAS). The evidence which supports the finding of a separate brain renin-angiotensin system (RAS) includes the following:
(a) the finding of a biosynthetic pathway for Ang II formation which includes Angiotensinogen and multiple enzymatic activities with the potential for forming angiotensin peptides; PA1 (b) neuronal sites where immunocytochemically identified Ang II has been localized; PA1 (c) the extraction of Ang II from brain tissue and its identification by high pressure liquid chromatography (HPLC); and, PA1 (d) angiotensin receptors demonstrated both by traditional membrane binding assay as well as by receptor autoradiography.
The discovery that neuronal elements in the brain produce Ang II has evinced Ang II's role as a regulatory neuropeptide in the central pathway subserving the maintenance of hydromineral balance and circulatory function. Although studies have shown that fragments containing the C-terminal sequence of Ang II mimic actions of the parent hormone in causing drinking (Fitzsimons, J. T., The Effect on Drinking of Peptide Precursors and of Shorter Chain Peptide Fragments of Angiotensin II Injected into the Rat's Diencephalon, J. Physiol. 214: 295-303, 1971; and Wright, J. W., Sullivan, M. J., Quirk, W. S., Batt, C. M. and Harding J. W., Heightened Blood Pressure and Drinking Responsiveness to Intracerebroventricularly Applied Angiotensins in the Spontaneously Hypertensive Rat, Brain Res. 420: 289-294, 1987), vasopressin (AVP) secretion (Fyhrquist, F., Eriksson, L., and Wallenius, M., Plasma Vasopressin in Conscious Goats After Cerebroventricular Infusions of Angiotensins, Sodium Chloride, and Fructose, Endocrinology 104: 1091-1095, 1979), increases in blood pressure (Fink, G. D. and Bruner, C. A., Hypertension During Chronic Peripheral and Central Infusion of Angiotensin III, Am. J. Physiol. 249: E201-E208, 1985; and Yang H. Y. T., Erdos, E. G., and Chiang, T. S., New Enzymatic Route for the Inactivation of Angiotensin, Nature 218: 1224-1226, 1986) and excitation of rat paraventricular (PVN) neurons (Harding, J. W., and Felix, D., Angiotensin-Sensitive Neurons in the Rat Paraventricular Nucleus: Relative Potencies of Angiotensin II and Angiotensin III, Brain Res. 410: 130-134, 1987), the view that Ang II is the active principle of the RAS has prevailed.
While the brain and peripheral renin-angiotensin systems are independent and kept apart by the blood-brain barrier (BBB), the two systems appear to be actively involved in the control of systemic blood pressure and the development and maintenance of hypertension. More particularly, in the central nervous system, Ang II may participate in the central regulation of blood pressure by augmenting sympathetic and parasympathetic efferent discharges, by the release of arginine vasopressin (AVP) and corticotropin releasing factor (CRF) and by stimulating thirst. It is generally thought that brain Ang II is synthesized in the supraoptic nucleus and paraventricular nuclei. Because of the connections of these nuclei to circumventricular organs and median preoptic area, there is a circuitry involving Ang II to produce increased vasopressin and sympathetic activity while simultaneously inhibiting the baroreflex. These three factors are thought to act in parallel to raise blood pressure.
Furthermore, in an attempt to regulate and/or control the blood pressure produced by the brain and/or peripheral renin-angiotensin systems, a number of enzyme inhibitors of the RAS, such as renin inhibitors (i.e. synthetic phosphatidyl ethanolamine) and converting enzyme inhibitors (i.e. captopril or teprotide) have been developed and introduced into hypertension therapy to reduce the production of Ang II by the RAS. In addition, receptor antagonists, such as antagonists for Ang II receptors (i.e. saralasin) have also been utilized in order to regulate the blood pressure of mammals.
The mechanism of action of the ACE inhibitors, such as the nonsulfahydryl converting enzyme inhibitors MK-421 and its active diacid form, MK-422, produced by Merck Sharpe and Dohme, U.S.A., is presumed to be the inhibition of angiotensin converting enzyme (ACE) in the RAS at the point where Ang I is converted to Ang II. However, recent evidence questions the overall effectiveness of the angiotensin converting enzyme (ACE) inhibitors in reducing hypertension. Moreover, since ACE has a peptidyldipeptidase action on several neuropeptidases which have also been found in the brain in addition to Ang II, i.e. bradykinin, enkephalin, and luteinizing releasing hormone, use of enzyme inhibitors such as MK-421 and MK-422 may not be specific for treatment of all hypertensions.
Notwithstanding the above, the present invention is directed to the use of Z-Pro-prolinal (ZPP), an inhibitor for proyl endopeptidase, for decreasing the rate of Ang-(1-7) inhibitors such as Ang-(1-7) and/or related N-terminal fragments production, thereby reducing and/or regulating hypertension. The present invention is based on the recent findings that (1) although it had been previously concluded that among the numerous analogs of Ang II the phenylalanine group at position 8 possesses the information for biological response and thus, Ang(1-7) was inactive, applicants lave recently discovered that Ang(1-7) is as potent as Ang II in stimulating vasopressin (AVP) secretion in the brain; and, (2) certain compositions such as ZPP, SCH 39, 370 and cFP-A-A-F-pAB are effective inhibitors of the formation of Ang-(1-7) from Ang I and/or Ang II; and (3) ZPP, when administered to genetic hypertensive mammals causes a significant decrease in mean arterial pressure which is unaccompanied with any significant changes in heart rate and other circulatory functions.